Longitudinal study on premature atherosclerosis in patients with systemic lupus erythematosus

Atherosclerosis 206 (2009) 546–550

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Longitudinal study on premature atherosclerosis in patients with systemic lupus erythematosus Karina de Leeuw a,∗ , Andries J. Smit b , Eric de Groot c , Arie M. van Roon b , Cees G. Kallenberg a , Marc Bijl a a b c

Department of Rheumatology & Clinical Immunology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Department of Vascular diseases, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Department of Vascular Medicine, Academic Medical Center, Amsterdam, The Netherlands

a r t i c l e

i n f o

Article history: Received 25 September 2008 Received in revised form 2 March 2009 Accepted 15 March 2009 Available online 25 March 2009 Keywords: Atherosclerosis Risk factors Systemic lupus erythematosus Intima–media thickness Longitudinal study

a b s t r a c t Objectives: To determine risk factors of accelerated atherosclerosis and progression of intima–media thickness (IMT) in patients with systemic lupus erythematosus (SLE). Methods: 74 SLE patients, age ranging from 13 to 69 years, and 74 age- and sex-matched controls were included. IMT of the common carotid artery was determined by B-mode ultrasound imaging. Traditional risk factors for atherosclerosis and disease-related factors were recorded. Cardiovascular risk was estimated using systematic coronary risk evaluation (SCORE). Markers of inflammation (C-reactive protein, CRP) and endothelial activation (thrombomodulin, vascular cell adhesion molecule-1, and von Willebrand factor) were determined. Measurements were repeated in 52 patients after a follow-up of 32 ± 7 months. Results: IMT was increased in SLE patients compared to controls. Prevalence of smoking and hypertension, use of lipid-lowering drugs and SCORE were higher in patients, as well as levels of CRP and markers of endothelial activation. The age-related increase in IMT was significantly higher in patients than in controls. In multivariate analysis, age and disease duration was independently related to IMT. Increase in IMT during follow-up was related to age only. Conclusion: The age-related increase in IMT is higher in SLE, indicating that atherosclerosis is accelerated in SLE patients. This is mainly due to disease-related risk factors, as disease duration was independently associated with IMT. © 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Development of atherosclerosis is accelerated in systemic lupus erythematosus (SLE). The cause of accelerated atherosclerosis in SLE remains unclear, as it has been demonstrated that traditional risk factors alone cannot explain the increased prevalence of atherosclerosis [1–6]. Therefore, additional, so-called nontraditional factors, such as systemic chronic inflammation, presence of auto-antibodies, enhanced endothelial cell activation and use of immunosuppressive drugs, have been suggested to contribute. Systemic chronic inflammation, manifested by increased levels of C-reactive protein (CRP), has been associated with atherogenesis [7]. As elevated levels of CRP have been demonstrated in SLE [8], systemic inflammation might enhance atherosclerosis in this disease. The contribution of autoimmunity to atherosclerosis is

∗ Corresponding author at: Department of Internal Medicine, Division of Clinical Immunology, University Medical Center Groningen, Hanzeplein 1, 9713 GZ or PO Box 30.001, 9700 RB, Groningen, The Netherlands. Tel.: +31 50 3614006; fax: +31 50 3619069. E-mail address: [email protected] (K. de Leeuw). 0021-9150/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2009.03.018

illustrated by the in vitro ability of auto-antibodies to activate endothelium [9], which is one of the earliest step in the development of atherosclerosis [7]. In vivo, endothelial cell activation has been demonstrated in SLE by the presence of increased levels of vascular cell adhesion molecule-1 (VCAM-1), thrombomodulin (TM) and von Willebrand factor (vWf) [10], and by increased expression of adhesion molecules in skin biopsy specimens of SLE patients [11]. Steroid treatment is often believed to be atherogenic, because its adverse effects, including hypertension, diabetes mellitus and dyslipidemia, are all risk factors for cardiovascular disease (CVD). However, steroid treatment could actually prevent atherosclerosis as well, because it suppresses inflammation, which is implicated in atherosclerosis. The results of studies about the effect of steroid treatment on atherosclerosis are conflicting, demonstrating a negative association [4], a positive association [12] or no association [2]. Detection of atherosclerosis at an early stage is important, as it may offer the possibility to identify patients who are at risk for CVD. Intima–media thickness (IMT) of the common carotid artery (CCA) measured by ultrasound is commonly accepted as a surrogate marker for subclinical atherosclerosis [13]. This non-invasive

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technique is well validated and often used in clinical practice. In fact, IMT has been shown to be associated not only with individual cardiovascular risk factors, but also with the development of CVD in the general population [14]. Most studies concerning IMT in SLE were cross-sectional in design [1–5]. The aim of the present study was to assess the prevalence and progression of IMT thickening in SLE patients. In addition, we investigated possible associations between progression and traditional and non-traditional risk factors for atherosclerosis. 2. Patients and methods 2.1. Patients Seventy-four consecutive patients fulfilling the American College of Rheumatology criteria for SLE [15], who attended the out-patient clinic of the University Medical Center Groningen and the Academic Medical Center Amsterdam, were included. Disease activity was assessed by the SLE disease activity index (SLEDAI) [16] and active disease was defined as SLEDAI > 4. Active disease within 4 months before participation was an exclusion criterion to prevent false positive IMT thickening due to active disease, as has been demonstrated in Takayasu’s arteritis [17] and rheumatoid arthritis [18]. Seventy-four subjects, either working in the hospital or who responded to an advertisement in the local newspaper, were recruited as controls. Exclusion criteria for controls were previously documented CVD or other diseases such as auto-immune diseases. The local research ethics committees gave approval for the study and written informed consent was obtained from each participant. Information was obtained regarding presence of CVD, defined as a history of ischemic heart disease (ICD-9 classification 410–414), cerebrovascular accidents or peripheral vascular disease based on medical records. Hypertension was defined as systolic arterial pressure above 140 mmHg and/or diastolic arterial pressure above 90 mmHg, or use of antihypertensive drugs, prescribed with the aim to reduce blood pressure [19]. Dyslipidemia was diagnosed if plasma cholesterol exceeded 6.21 mmol/l, plasma LDL cholesterol exceeded 3.36 mmol/l, plasma triglycerides exceeded 2.26 mmol/l, or when the patients used HMG-CoA inhibitors [19]. Furthermore, body mass index (BMI), smoking status, diabetes and family history of CVD (considered positive if first-degree relatives suffered from CVD before 60 years of age) were recorded. To obtain an estimation of the overall cardiovascular risk the systematic coronary risk evaluation (SCORE) risk analysis was used [20]. This estimation of 10-year risk of fatal CVD is based on gender, age, total cholesterol level, systolic blood pressure and smoking status. As persons aged 30 are essentially risk free within the next 10 years, using the SCORE adjusted for that age would give a wrong impression of the long-term risk for young people with high number of risk factors. The cardiovascular risk estimation was, therefore, extrapolated to the age of 60 years to circumvent such an underestimation. Furthermore, we assessed disease-related factors which might influence the development of atherosclerosis. Next to disease duration, disease-related damage was determined at the time of enrolment in the study using the Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index (SLICC/ACR DI) [21]. Also, SLICC/ACR DI corrected for damage due to CVD was recorded. The use of corticosteroids, hydroxychloroquine and azathioprine was assessed, including present use, daily dose and cumulative dose. All measurements, including traditional and non-traditional risk factors and IMT, were repeated in 52 patients of whom data were available after a follow-up period of at least 20 months. In the other 22 patients, follow-up measurements were not performed because of a follow-up period < 20

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months (n = 12), three patients had moved, three patients withdrew informed consent and in four patients IMT measurements were inadequate due to technical problems. 2.2. Measurements of IMT To measure the extent of atherosclerosis, we used ultrasound of the far wall of the CCA as described before [22]. This segment can be assessed with high reproducibility [23]. Mean IMT (m-IMT; the mean of the segment studied) and maximum IMT (M-IMT; the highest IMT value found among the segment studied) were calculated. Plaque was defined as a maximum IMT > 1 mm. Progression was defined as an increase in IMT > 0.03 mm, as the coefficient of variation is approximately 5%, and mean IMT in this study was 0.6 mm [23]. 2.3. Blood analyses Lipid levels were measured by routine techniques. Additional samples for measuring levels of markers of endothelial activation and inflammation were stored at −20 ◦ C until analysis. Serum levels of VCAM-1 (R&D Systems, Abingdon, UK) and TM (Diaclone, Besanc¸on, France) were measured according to the manufacturer’s instructions. vWf and CRP were determined using in-house enzyme-linked immunosorbent assays as described before [22]. 2.4. Statistical methods Except when stated otherwise, values are expressed as mean ± standard deviation, when variables were normally distributed. In case of a non-normal distribution data are reported as median (interquartile range). Comparisons between patients and controls and between the different groups of patients were made by two-sample t-tests or Mann–Whitney tests for continuous variables and by chi-square analysis for categorical variables. The univariate correlation between IMT and all included variables was assessed by Pearson correlation coefficient. Multiple linear regression analysis was performed with mean IMT as the dependent variable and all variables which were significantly correlated in univariate analyses as independent variables. Disease-related variables were transformed into dummy variables to perform this multivariate analysis using data of controls and patients. Comparisons between variables at evaluation 1 and evaluation 2 of SLE patients were made by paired-samples t-test or Wilcoxon signed ranks test for continuous variables and by McNemar analysis for categorical variables. Also, multiple linear regression analysis was performed with mean IMT at evaluation 2 as the dependent variable and all variables which showed a correlation with a p-value > 0.1 in univariate analyses as independent variables. All analyses were performed using SPSS 12.0. A two-sided pvalue < 0.05 was considered to indicate statistical significance. 3. Results 3.1. Characteristics In SLE, smoking and hypertension were more prevalent compared to controls (Table 1). Also, the use of antihypertensive drugs and lipid-lowering drugs, prescribed because of hypercholesterolemia, was increased in patients. Levels of cholesterol and HDL were decreased in patients. In addition, combining conventional risk factors using the SCORE analysis, showed that SLE patients had an increased risk for fatal CVD in the next 10 years. Diseaserelated factors of the patients are shown in Table 2. In this cohort, nine patients had manifest CVD. Patients with CVD tended to be older than those without CVD (46 ± 10 years vs. 36 ± 15 years,

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Table 1 Traditional risk factors for CVD in patients and controls. Patients (n = 74) Age (years) Men, n (%) BMI (kg/m2 ) Smokers, n (%) Hypertension, n (%) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Use of antihypertensives, n (%)

p = 0.07). Other traditional risk factors did not differ between both groups. Controls (n = 74)

37 ± 14 11 (15) 25 ± 4 18 (24)

38 ± 14 8 (11) 24 ± 3 7 (9)

23 (31) 120 ± 15 75 ± 9 20 (27)

5 (7) 111 ± 14 72 ± 9 3 (4)

p

3.2. Endothelial activation markers

NS NS NS 0.03 <0.001 <0.001 0.02 <0.001

Hyperlipidemia, n (%) Use of lipid-lowering drugs, n (%) Increased lipid levels, n (%) Cholesterol (mmol/l) HDL (mmol/l) LDL (mmol/l)

30 (41) 9 (12) 22 (30) 4.8 ± 1.0 1.1 ± 0.4 3.1 ± 0.9

28 (38) 0 28 (38) 5.4 ± 1.0 1.4 ± 0.5 3.6 ± 1.0

NS <0.01 NS 0.02 0.02 0.08

Diabetes mellitus, n (%) Family history for CVD, n (%) SCORE extrapolated to 60 years (% in next 10 years)

4 (5) 21 (28) 2.2 (1.7–4.1)

0 14 (19) 1.8 (1.5–2.4)

NS NS 0.01

3.3. Intima–media thickness

Unless otherwise indicated, data are expressed as mean ± standard deviation when normally distributed and as median (25–75%) when non-normally distributed; CVD: cardiovascular disease, BMI: body mass index, and SCORE: systematic coronary risk evaluation.

Table 2 Disease characteristics of SLE patients. Patients, n (%) Disease duration (months) SLEDAI SLICC/ACR DI

98 (33–178) 2 (0–2) 0 (0–1)

ACR criteria Malar rash Discoid rash Photosensitivity Oral ulcers Arthritis Serositis Renal disorder Neurologic disorder Haematologic disorder Immunologic disorder Antinuclear antibody

38 (51) 24 (32) 32 (43) 13 (18) 42 (57) 29 (39) 34 (46) 5 (7) 49 (66) 63 (85) 74 (100)

Prednisolone use, n (%) Daily mean dose (mg)

33 (45) 7.5 (5–10)

Hydroxychloroquine use, n (%) Daily mean dose (mg)

29 (39) 400 (200–400)

Azathioprine use, n (%) Daily mean dose (mg)

15 (20) 125 (100–150)

SLEDAI: SLE disease activity score and SLICC/ACR DI: systemic lupus international collaborating clinics/American College of Rheumatology damage index.

Endothelial activation markers were measured to investigate whether endothelial activation was present in SLE patients during inactive disease. Levels of VCAM-1 and vWf were elevated in SLE compared to controls, 446 (361–555) ng/ml vs. 352 (292–400) ng/ml (p < 0.001), and 152 (114–233)% vs. 83 (56–124)% (p < 0.001), respectively. Also, plasma levels of CRP were increased in SLE patients, 1.8 (0.7–6.4) mg/l vs. 0.7 (0.3–2.4) mg/l (p = 0.001). Levels of TM did not differ between both groups: 2.7 (2.0–3.7) ng/ml vs. 2.7 (1.9–3.9) ng/ml.

Overall, SLE patients had an increased mean IMT (Table 3). Stratifying the group for age revealed that in particular after the age of 30 years, mean IMT and maximum IMT of SLE patients were significantly elevated compared to controls. Plaques were present in five patients (7%) and one control (1%) (p = 0.1). Comparing patients with and without CVD, IMT was increased in SLE patients with CVD (mean IMT: 0.68 (0.63–0.77) mm vs. 0.60 (0.55–0.68) mm, p = 0.02, and maximum IMT: 0.82 (0.74–0.91) mm vs. 0.73 (0.66–0.81) mm, p = 0.03). All significant univariate correlations between mean as well as maximum IMT and included risk factors are shown in Table 4. All other investigated risk factors, including gender, BMI, smoking, lipid levels, diabetes mellitus, family history for CVD, levels of other endothelial activation markers, SLEDAI, SLICC, medication (both use, as well as the daily mean dose), did not correlated to IMT in univariate analsyses. The correlation between age and IMT is depicted in Fig. 1. A significantly steeper regression line between age and IMT is shown in SLE patients compared to controls. Furthermore, this figure demonstrates that patients younger than 20 years old, who were included at the time of diagnosis, do not differ in IMT compared to age- and sex-matched controls. The relation between IMT, disease duration and age in SLE is shown in Fig. 2, demonstrating that age and disease duration are related to mean IMT. This was confirmed in multivariate analyses, in which age (B = 0.004, p < 0.001) and duration >10 years (B = 0.054, p = 0.01) were independently correlated to mean IMT. 3.4. Follow-up of IMT measurement in SLE patients In 52 SLE patients, systolic and diastolic blood pressure significantly increased after follow-up of 32 ± 7 months (119 ± 16 mmHg vs. 128 ± 15 mmHg, p = 0.001, and 76 ± 10 mmHg vs. 81 ± 9 mmHg, p = 0.003, respectively). No changes in other risk factors, including use of antihypertensive drugs, were found. Levels of VCAM-1 decreased, 475 (362–620) ng/ml vs. 374 (275–486) ng/ml (p = 0.01),

Table 3 IMT measurements in patients and controls. Patients

Controls

p-value

All (n = 74)

m-IMT (mm) M-IMT (mm)

0.61 (0.56–0.68) 0.73 (0.66–0.82)

0.58 (0.54–0.65) 0.73 (0.66–0.77)

0.05 0.14

Age <30 years (n = 23)

m-IMT (mm) M-IMT (mm)

0.51 (0.42–0.59) 0.62 (0.49–0.70)

0.56 (0.47–0.60) 0.67 (0.55–0.73)

0.25 0.19

Age 30–45 years (n = 30)

m-IMT (mm) M-IMT (mm)

0.63 (0.58–0.68) 0.74 (0.73–0.81)

0.56 (0.53–0.65) 0.69 (0.62–0.77)

0.002 0.008

Age >45 years (n = 21)

m-IMT (mm) M-IMT (mm)

0.68 (0.63–0.87) 0.85 (0.77–0.98)

0.62 (0.58–0.72) 0.73 (0.70–0.88)

0.017 0.050

Data are expressed as median (25–75%); IMT: intima–media thickness, m-IMT: mean IMT, and M-IMT: maximum IMT.

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Table 4 Univariate regression analysis between IMT and risk factors for CVD, showing significant correlations only. Variable

m-IMT

M-IMT

Patients (n = 74)

Controls (n = 74)

r

p

r

Age

0.77

<0.001

Blood pressure Systolic Diastolic

0.32 0.26

0.01 0.03

vWf SCORE Disease duration

0.37 0.32 0.43

0.003 0.01 <0.001

Patients (n = 74)

Controls (n = 74)

p

r

p

0.62

<0.001

0.75

<0.001

0.62

<0.001

0.40 0.29

0.001 0.001

0.40 0.31

<0.001 0.01

0.35 0.29

0.002 0.01

NS 0.02 NA

0.32 0.37 0.39

0.01 0.002 0.001

−0.20 0.23

−0.09 0.33

r

p

NS NS NA

CVD: cardiovascular disease, m-IMT: mean IMT, M-IMT: maximum IMT, vWf: von Willebrand factor, SCORE: systematic coronary risk evaluation, NS: not significant, and NA: not applicable.

Fig. 1. Correlation between intima–media thickness and age in SLE patients and controls. In SLE patients and controls (CTL), intima–media thickness (IMT) is positively correlated with age. Open circles represent controls and closed circles represent SLE patients. The dotted line represents the correlation between IMT and age in controls (r = 0.62, p < 0.001). The straight line represents the correlation between IMT and age in patients (r = 0.77, p < 0.001). The slope is steeper in SLE compared to controls (p < 0.01).

Fig. 2. Association between intima–media thickness, age, and disease duration in SLE. Mean IMT according to tertiles of age (<30, 30–45 and >45 years) and tertiles of disease duration (<5, 5–10 and >10 years) in SLE patients.

whereas levels of TM and CRP increased during follow-up, 2.6 (2.0–3.7) ng/ml vs. 3.2 (2.4–4.7) ng/ml (p = 0.01), and 1.0 (0.5–5.5) mg/l vs. 1.9 (0.9–6.7) mg/l (p = 0.03), respectively. Mean and maximum IMT at evaluation 2 were correlated to age (r = 0.54, p < 0.001, and r = 0.61, p < 0.001, respectively) and to systolic blood pressure (r = 0.38, p = 0.005 and r = 0.33, p = 0.02, respectively). Other variables were not significantly correlated to IMT at evaluation 2. Mean IMT tended to be increased and maximum IMT was increased compared to evaluation 1 (0.63 ± 0.10 mm vs. 0.66 ± 0.15 mm (p = 0.06), and 0.75 ± 0.12 mm vs. 0.80 ± 0.19 mm (p = 0.02), respectively). Mean progression of IMT was 0.012 ± 0.04 mm/year. Among 52 patients studied, 31 (60%) had no increase in mean IMT, and 21 (40%) had progression of mean IMT, defined as a change of IMT > 0.03 mm. Baseline findings of patients with and without progression were similar (data not shown). To assess which variable could predict the change in IMT, univariate analyses were performed, correlating all traditional and non-traditional variables at evaluation 1 to mean IMT at evaluation 2. In patients with progression of IMT, age (r = 0.53, p = 0.01) was correlated to IMT at evaluation 2, which was confirmed in multivariate analysis (data not shown). 4. Discussion Atherosclerosis is a major cause of morbidity and mortality in SLE. In this study, we demonstrated that increased IMT, as a measure of atherosclerosis, is more prevalent in SLE patients compared to controls. More importantly, we clearly showed that this higher prevalence is for a major part SLE dependent, as disease duration was independently correlated to IMT. We also found a stronger age-related increase of IMT in patients compared to controls. This pattern is similar to that found in patients with familial hypercholesterolemia (FH) [24]. Like in FH, IMT values in young SLE patients were not different from those in age-matched controls, whereas with increasing age, differences in IMT became apparent between patients and controls. After a follow-up of 32 ± 7 months, we found a mean progression in IMT of 0.012 ± 0.04 mm/year in SLE patients. In a large population study including healthy men and women aged 45–64 years, Howard et al. estimated the average rate of progression of IMT in the CCA as 0.009 mm/year in Caucasian women [25]. Mackinnon et al. even found a much lower progression of mean IMT of the CCA, as in this German primary health care service population progression of IMT was 0.001 mm/year [26]. Compared to these data, progression in IMT over time in SLE is significantly increased. These findings are in line with the study of Roman et al. [6]. Which SLE dependent features are involved is still unknown. Among others, we speculate that during the course of the disease, disease-related factors promote endothelial activation, which eventually leads to atherosclerosis [1–4]. In this study, we indeed

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demonstrated that, besides traditional risk factors, disease duration was correlated to IMT. Until now, only two longitudinal studies have been reported [5,6]. Doria et al. described a follow-up study in which IMT was measured after a follow-up of 5 years [5]. They found that IMT had progressed in patients who had at baseline active renal disease and active disease. Roman et al. performed a longitudinal study in which progression of atherosclerosis was measured in 158 SLE patients after a follow-up of 34 ± 9 months. Progression of atherosclerosis was independently associated with older age at diagnosis, longer duration of SLE, and higher homocystein concentrations [6]. In our follow-up study, progression of IMT, occurred in 21 out of the 52 (40%) patients. No differences in baseline findings were found comparing patients with and without progression. Furthermore, except for age, no other predictors of progression of IMT could be determined. Several explanations can be given why, in contrast to Roman et al., no predictive risk factors were found. First, Roman et al. determined plaque score, defined as the number of plaques in different segments including CCA, bulb, and internal carotid artery at both sites, whereas we measured IMT at one site, namely in the left CCA. The reproducibility of measuring IMT at this segment has been described as rather high [23]. However, as atherosclerosis appears later in the CCA than in the bulb or internal carotid artery, which are included in the plaque score used by Roman et al., the prevalence of atherosclerosis and increased IMT in our patients might be underestimated [13]. It is still unclear whether measuring IMT in the CCA only is a better indicator to predict CVD than measuring IMT at different sites, resulting in a mean score [27]. Secondly, our patients were predominantly Caucasians (95%) compared to 48% of the patients included by Roman et al. In general, Caucasians with SLE have milder disease than other ethnicities and, therefore, a lower prevalence of atherosclerosis might be expected, as shown by the association of IMT and damage index. Furthermore, we included a smaller number of patients. The latter might also explain why in the current study no relation between CRP and endothelial activation markers, such as VCAM-1, and atherosclerosis was found. Previously, CRP has been shown to be an independent prognostic marker for CVD [28], and levels of VCAM-1 have been demonstrated to be independent predictors of long-term clinical outcomes, including CVD [29]. Furthermore, in the present study CRP (and other markers) was only measured at one time point, whereas a cumulative CRP value is probably a better reflection of the exposure to inflammation during a longer period. Therefore, to be able to investigate the hypothesis that chronic inflammation and endothelial activation influence progression of atherosclerosis in SLE, further studies including more patients for a longer period should be performed. Nevertheless, even in our small cohort of 52 patients, the progression of IMT in SLE patients is increased compared to healthy controls as measured in large population studies. In conclusion, this study demonstrates that the accelerated atherosclerosis found in SLE patients is SLE dependent, and, therefore, emphasizes the need for interventions to modify progression of atherosclerosis at an early time point after the onset of the disease. References [1] Esdaile JM, Abrahamowicz M, Grodzicky T, et al. Traditional Framingham risk factors fail to fully account for accelerated atherosclerosis in systemic lupus erythematosus. Arthritis Rheum 2001;44:2331–7. [2] Asanuma Y, Oeser A, Shintani AK, et al. Premature coronary-artery atherosclerosis in systemic lupus erythematosus. N Engl J Med 2003;349:2407–15. [3] Leeuw K, de, Freire B, Smit AJ, Bootsma H, Kallenberg CG, Bijl M. Traditional and non-traditional risk factors contribute to the development of acceler-

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